In the last few years the study of the biology of Trypanosoma brucei, the agent of sleeping sickness in humans and nagana in cattle, has witnessed an incredible growth due to two major advances: the completion of the T. brucei genome sequence and the discovery by our laboratory of RNA interference (RNAi). By interfacing genome information with down-regulating gene function by RNAi, it is now realistic to investigate complex cellular mechanisms using global approaches. As transcription regulation plays a minor role in T. brucei, the emphasis on regulation of gene expression is primarily on post-transcriptional regulatory mechanisms. Thus, we anticipate that the enzymes governing mRNA metabolism are essential for these parasites. This proposal focuses on aspects of RNA metabolism that are either unique to trypanosomes (cap 4 biosynthesis and function) or are novel concepts in eukaryotic biology (coupling of trans-splicing and polyadenylation and the RNAi pathway). The long-term goal of this proposal remains the characterization of molecular mechanisms involved in RNA metabolism and their relevance to trypanosome biology. In the next funding period we plan to: 1. Characterize the specific RNA methyltransferases involved in cap 4 biosynthesis and investigate whether there is a mechanistic link between cap 4 formation and polyadenylation. 2. Further our understanding of the coupling of trans-splicing and polyadenylation by using a variety of approaches, including monitoring the association of processing factors along transcribed genes in living cells. 3. Further characterize the RNAi pathway in terms of its components and how the interaction of TbAGO1 with translating ribosomes is brought about.